1. Field of the Invention
This invention relates to a recombinant interleukin-2 composition and a process for preparing it.
2. References
Interleukin-2 (IL-2) is a soluble protein which is capable of modulating lymphocyte reactivity and promoting the long-term in vitro culture of antigen-specific effector T-lymphocytes and, in the past, has been produced by stimulating mouse, rat or human lymphocyte cells with a mitogen. Human IL-2 consists of a 133 amino acid polypeptide containing a single intramolecular disulfide bridge.
U.S. Pat. No. 4,401,756, issued to Gillis on August 30, 1983, discloses a process for preparing IL-2 from malignant cells by culturing human leukemia or lymphoma cells in a serum containing medium supplemented with various additives. The culture is stimulated by an optimum concentration of T-cell mitogen to produce a supernate which contains IL-2. After a period of time, the supernate is collected and processed to purify the IL-2. The patent discloses that a cell line designated Jurkat-FHCRC is a preferred source of leukemic human T-cells.
U.S. Pat. No. 4,490,289, issued to Stern on December 25, 1985, discloses a process for purifying human IL-2 derived from induced human malignant cells. The IL-2 is purified to homogeneity by using multiple steps of reverse phase high performance liquid chromatography. The patent discloses that the purified IL-2 exhibits potent activity promoting the long-term in vitro culture of antigen-specific effector T-lymphocytes and in modulating lymphocyte activity. The Jurkat-FHCRC leukemic human T cell line is said to be a preferred cell line and the resulting purified material is disclosed in one example as having an activity of 205,000 units/ml and in another example of 983,040 units/ml. The patent mentions a prior art preparation of IL-2 from normal lymphocytes which was unstable even at -70.degree. C. and required bovine serum albumin (BSA) or polyethylene glycol to retain activity. One unit of activity is defined in the patent as the number of microliters present in a T cell culture well which induced 50% of maximal thymidine incorporation.
Urdal, et al., Journal of Chromatography, 296, 171-179 (1984), disclose the purification of human IL-2 (Jurkat) on a C.sub.8 reverse-phase column in pyridine-acetate-propanol followed by chromatography on a C.sub.18 reverse-phase column in trifluoroacetic acid-acetonitrile.
Taniguchi, et al., Nature, 302, 305 (1983), disclose the isolation of a human IL-2 complementary DNA clone from the Jurkat cell line and the determination of its nucleotide sequence.
Rosenberg, et al., Science, 223, 1412-1415 (1984), disclose the isolation of the gene for interleukin-2 from the Jurkat cell line and from normal peripheral blood lymphocytes, insertion of the gene into Escherichia coli, and expression of the gene by the resulting transformed microorganism. The IL-2 was purified to apparent homogeneity. The authors reported that no functional differences between native and recombinant IL-2 molecules were detected.
European patent application No. 84304992.5, European publication No. 0 133 767 A2, discloses that gamma interferon obtainable from the human leukocytes, which is unstable even during lyophilization and storage in solid state, can be stabilized by addition of albumin and/or a sugar. Usable sugars are said to include monosaccharides, disaccharides, sugar alcohols and mixtures thereof. Specifically mentioned are compounds such as glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol and xylitol. The publication also discloses that a stable, lyophilized gamma interferon composition is prepared by lyophilizing its aqueous solution containing this stabilizer without lowering its activity.
Gekko, et al., Biochemistry 20, 4677-4686 (1981) discuss a thermodynamic and kinetic study of protein stabilization by glycerol. The particular proteins involved were chymotrypsinogen and ribonuclease.
When recombinant IL-2 (rIL-2) is produced from transformed E. coli, the rIL-2 is recovered and purified in a post-fermentation process which involves sonicating the cell paste, extracting the useful protein, purifying the extracted rIL-2 by use of reverse phase high performance liquid chromatography (HPLC), and resuspending the resulting purified rIL-2 in water often with a carrier such as human serum albumin. If the rIL-2 is not going to be used immediately, generally it is lyophilized after HPLC purification and resuspended when needed. The specific activity of this resuspended rIL-2 composition is typically below about 50,000 units/mg of protein, which is about 16.7% of that of pure human Jurkat IL-2, but may be as high as about 90,000 units/mg.
When a protein such as IL-2 is to be used for therapeutic purposes, it is desirable that the specific activity be as high as possible, thereby minimizing concern about possible detrimental effects of the inactive protein. Moreover, recently there has been considerable public concern about using any biological products having a component derived from human blood. This concern has most recently been centered on plasma-derived factor VIII and natural human growth hormone but could include components such as human serum albumin. Thus, a rIL-2 composition with high specific activity and lacking any carrier derived from human blood is highly desirable.